Upflow hydrolysis reactor system for hydrolyzing cellulose

ABSTRACT

A process for the enzymatic hydrolysis of cellulose to produce a hydrolysis product from a pre-treated cellulosic feedstock is provided. The process comprises introducing an aqueous slurry of the pre-treated cellulosic feedstock at the bottom of a hydrolysis reactor. Axial dispersion in the reactor is limited by avoiding mixing and maintaining an average slurry flow velocity of about 0.1 to about 20 feet per hour, such that the undissolved solids flow upward at a rate slower than that of the liquid. Cellulase enzymes are added to the aqueous slurry before or during the step of introducing. An aqueous stream comprising hydrolysis product and unhydrolyzed solids is removed from the hydrolysis reactor. Also provided are enzyme compositions which comprise cellulase enzymes and flocculents for use in the process. In addition, a kit comprising cellulase enzymes and flocculent is provided.

The present invention relates to processes for the conversion ofcellulosic feedstocks. More specifically, the present invention relatesto processes for enzymatic conversion of cellulosic feedstocks havingimproved efficiency.

BACKGROUND OF THE INVENTION

Fuel ethanol is currently produced from feedstocks such as cornstarch,sugar cane, and sugar beets. However, the production of ethanol fromthese sources cannot expand much further due to limited farmlandsuitable for the production of such crops and competing interests withthe human and animal food chain. Finally, the use of fossil fuels, withthe associated release of carbon dioxide and other products, in theconversion process is a negative environmental impact of the use ofthese feedstocks

The possibility of producing ethanol from cellulose-containingfeedstocks such as agricultural wastes, grasses, and forestry wastes hasreceived much attention due to the availability of large amounts ofthese inexpensive feedstocks, the desirability to avoid burning orlandfilling cellulosic waste materials, and the cleanliness of ethanolas a fuel compared to gasoline. In addition, a byproduct of thecellulose conversion process, lignin, can be used as a fuel to power thecellulose conversion process, thereby avoiding the use of fossil fuels.Studies have shown that, taking the entire cycle into account, the useof ethanol produced from cellulose generates close to nil greenhousegases.

The cellulosic feedstocks that may be used for ethanol productioninclude (1) agricultural wastes such as corn stover, wheat straw, barleystraw, oat straw, oat hulls, canola straw, and soybean stover; (2)grasses such as switch grass, miscanthus, cord grass, and reed canarygrass; (3) forestry wastes such as aspen wood and sawdust; and (4) sugarprocessing residues such as bagasse and beet pulp.

Cellulose consists of a crystalline structure that is very resistant tobreakdown, as is hemicellulose, the second most prevalent component ofcellulosic feedstocks. The conversion of cellulosic fibers to ethanolrequires: 1) liberating cellulose and hemicellulose from lignin orincreasing the accessibility of cellulose and hemicellulose within thecellulosic feedstock to cellulase enzymes, 2) depolymerizinghemicellulose and cellulose carbohydrate polymers to free sugars, and 3)fermenting the mixed hexose and pentose sugars to ethanol.

Among well-known methods used to convert cellulose to sugars is an acidhydrolysis process involving the use of steam and acid at a temperature,acid concentration and length of time sufficient to hydrolyze thecellulose to glucose (Grethlein, 1978, J. Appl. Chem. Biotechnol.28:296-308). The glucose is then fermented to ethanol using yeast, andthe ethanol is recovered and purified by distillation.

An alternative method of cellulose hydrolysis is an acid prehydrolysis(or pre-treatment) followed by enzymatic hydrolysis. In this sequence,the cellulosic material is first pre-treated using the acid hydrolysisprocess described above, but at milder temperatures, acid concentrationand treatment time. This pre-treatment process increases theaccessibility of cellulose within the cellulosic fibers for subsequentenzymatic conversion steps, but results in little conversion of thecellulose to glucose itself. In the next step, the pre-treated feedstockis adjusted to an appropriate temperature and pH, then submitted toenzymatic conversion by cellulase enzymes.

The hydrolysis of the cellulose, whether by acid or by cellulaseenzymes, is followed by the fermentation of the sugar to ethanol, whichis then recovered by distillation.

The efficient conversion of cellulose from cellulosic material intosugars, and the subsequent fermentation of sugars to ethanol, is facedwith a major challenge regarding commercial viability. In particular,acid prehydrolysis requires large amounts of acid. For a cleanfeedstock, such as washed hardwood, the sulfuric acid demand is 0.5% to1% of the dry weight of the feedstock; for agricultural fibers, whichcan contain high levels of silica, salts, and alkali potassium compoundsfrom the soil, the acid demand can be up to about 10-fold higher,reaching 5% to 7% by weight of feedstock. This adds significant cost tothe process. A second drawback of using large amounts of acids in aprehydrolysis process is that the acidified feedstock must beneutralized to a pH between about 4.5 and about 5 prior to enzymatichydrolysis with cellulase enzyme. The amount of caustic soda used toneutralize acidified feedstock is proportional to the amount of acidused to acidify the feedstock. Thus, high acid usage results in highcaustic soda usage, which further increases the cost of processingcellulosic feedstock to ethanol. Furthermore, the cost of enzymatichydrolysis is high, as cellulose remains resistant to hydrolysis despitepre-treatment, which increases the enzyme dosage required. Suchincreased requirement can be counteracted by increasing the hydrolysistimes (90-200 hours), in turn requiring very large reactors, which againadds to the overall cost.

A method of decreasing the enzyme dosage while maintaining high levelsof cellulose conversion is Simultaneous Saccharification andFermentation (SSF). In this type of system, enzymatic hydrolysis iscarried out concurrently with yeast fermentation of glucose to ethanolin a reactor vessel. During SSF, the yeast removes glucose from thereactor by fermenting it to ethanol and this decreases inhibition of thecellulase by glucose. However, the cellulase enzymes are still inhibitedby ethanol. SSF is typically carried out at temperatures of 35-38° C.,which is a compromise between the 50° C. optimum for cellulase and the28° C. optimum for yeast. This intermediate temperature leads tosubstandard performance by both the cellulase enzymes and the yeast.Thus, the inefficient catalysis requires very long reaction times andvery large reaction vessels—both of which are costly.

A method for higher volumetric productivity is disclosed in U.S. Pat.No. 5,258,293 (Lynd). This method utilizes a lignocellulosic feedstockalong with microorganisms that are continuously introduced into areaction vessel. Fluid is also continuously added from the bottom of thereaction vessel, but no mechanical agitation of the slurry occurs. Asthe reaction progresses, the lignocellulosic feedstock being digestedtends to accumulate in a spatially non-homogenous layer while theethanol product rises to a top layer, where it is removed. The insolublesubstrate accumulates in a bottom layer and can be withdrawn from thevessel. This arrangement results in a differential retention of thefermenting substrate, which allows for increased residence time in thereactor vessel.

In another approach, disclosed in U.S. Pat. No. 5,837,506 (Lynd),ethanol is produced using an intermittently agitated, perpetually fedbioreactor. Lignocellulosic slurry and microorganisms are added to areactor; the mixture is then agitated, either by mechanical means or byfluid recirculation, for a specific time interval, after which it isallowed to settle. Ethanol is then removed from a top portion of thereactor, additional substrate is added, and the cycle continues. In asimilar method, Kleijntjens et al. (1986, Biotechnology Letters,8:667-672) utilize an upflow reactor to ferment cellulose-containingsubstrate in the presence of C. thermocellum. The substrate slurrysettles to form an aggregated fibre bed, which is accelerated by slowmechanical stirring. Substrate is added periodically, while liquid iscontinuously fed to the reactor. Ethanol product accumulates in a toplayer, where it is removed from the reactor. The methods disclosed inU.S. Pat. No. 5,837,506, U.S. Pat. No. 5,258,293 and Kleijntjens et al.result in an increase in the residence time of the feedstock in thereactor vessel. However, all three methods suffer from the disadvantagesof the SSF process.

U.S. Pat. No. 5,348,871; U.S. Pat. No. 5,508,183; U.S. Pat. No.5,248,484; and U.S. Pat. No. 5,637,502 (Scott) teach a method to improvethe conversion efficiency in enzymatic hydrolysis through the use of anattritor in association with an agitated reactor vessel. The agitatorproduces a high-shear field for size reduction of solid particles in thecellulosic feedstock, which constantly provides new surface area for thecellulase enzymes. Therefore, the reaction efficiency is increased andthe enzyme requirements are decreased. However, the high shear ofteninactivates the enzymes. Furthermore, the cost of the attritor equipmentis much greater than the savings due to the decreased enzyme dosage.

U.S. Pat. No. 5,888,806 and U.S. Pat. No. 5,733,758 (Nguyen) teach analternative approach using a tower hydrolysis reactor comprisingalternating mixed and unmixed zones, thus reducing the mixing powerconsumption and cost. The slurry is moved upward in plug flow throughthe reactor and is intermittently mixed in the mixing zones, thuspreventing channelling of liquid and ensuring uniform heat and masstransfer. While the methods disclosed in U.S. Pat. No. 5,888,806 andU.S. Pat. No. 5,733,758 reduce the shearing and denaturation of theenzymes, the cost of the mixing equipment is substantial. Furthermore,the kinetic performance of the enzymes is no better than can be achievedin a batch hydrolysis mode.

At present there is much difficulty in the art to attain high conversionefficiency while maintaining lowered costs. Increasing hydrolysis timesto avoid higher costs of increasing the enzyme dosage requires largerreactors, which in turn increases equipment costs. Mixing andintermittent mixing of the feedstock during hydrolysis can increaseenzyme efficiency but equipment costs will again increase, and shearforces will cause enzyme denaturation. Other systems compromise theoptimal enzyme activity and reduce the efficiency of the enzymes.

SUMMARY OF THE INVENTION

The present invention relates to processes for the conversion ofcellulosic feedstocks into products. More specifically, the presentinvention relates to processes for the enzymatic conversion ofcellulosic feedstocks having improved efficiency.

According to the present invention, there is provided an upflow settlingreactor for enzymatic hydrolysis of cellulose.

The present invention also provides a process for the enzymatichydrolysis of cellulose to produce a hydrolysis product from apre-treated cellulosic feedstock, the process comprising:

i) providing an aqueous slurry of the pre-treated cellulosic feedstock,the slurry comprising from about 3% to about 30% undissolved solids in aliquid, the undissolved solids comprising at least about 20% cellulose;

ii) introducing the aqueous slurry at the bottom of a hydrolysis reactorand limiting axial dispersion in the reactor by avoiding mixing, andmaintaining an average slurry flow velocity of about 0.1 to about 20feet per hour, such that the undissolved solids flow upward at a rateslower than that of the liquid;

iii) adding cellulase enzymes to the aqueous slurry before or during thestep of introducing (step ii); and

iv) removing an aqueous stream comprising hydrolysis product andunhydrolyzed solids from the hydrolysis reactor, the hydrolysis productcomprising glucose, cellobiose, glucose oligomers, or a combinationthereof.

The present invention relates to the process for the enzymatichydrolysis of cellulose as defined above, wherein, in the step ofintroducing (step ii), the aqueous slurry is introduced at the bottom ofthe hydrolysis reactor with a uniform radial distribution.

The present invention is directed to the process for the enzymatichydrolysis of cellulose as defined above, wherein, in the step of adding(step iii), one or more than one flocculating compound is added to theaqueous slurry, separately from, or together with the cellulase enzymes,or a combination thereof. Furthermore, the one or more than oneflocculating compound may be added before or during the step ofintroducing (step ii), or a combination thereof.

The present invention pertains to the process for the enzymatichydrolysis of cellulose as defined above, wherein, in the step ofproviding (step i), the slurry comprises from about 5% to about 20% byweight undissolved solids, and the undissolved solids comprise fromabout 25% to about 70% by weight cellulose.

The present invention is directed to the process as described above,wherein the pre-treated cellulosic feedstock is obtained from wheatstraw, oat straw, barley straw, corn stover, soybean stover, canolastraw, sugar cane bagasse, switch grass, reed canary grass, cord grass,oat hulls, sugar beet pulp or miscanthus. Furthermore, the pre-treatedcellulosic feedstock may have been subjected to pre-treatment from about160° C. to about 280° C. and for about 3 seconds to about 30 minutes atan acid concentration from about 0% to about 5% prior to enzymatichydrolysis. The acid may be selected from the group consisting ofsulfuric acid, sulfurous acid, and sulfur dioxide. Optionally, a liquidstream comprising sugar may be separated from the pre-treated cellulosicfeedstock prior to the step of introducing (step ii). The liquid streammay be separated from the feedstock using processes such as filtration,centrifugation, washing or any other suitable process as would be knownin the art. If washing is used for the separation, it may be carried outusing a suitable washing medium such as water, a recycled processstream, treated effluent, or a combination thereof.

The present invention also provides for the process as described above,wherein, in the step of adding (step iii), the cellulase enzyme is addedat a dosage from about 1.0 to about 40.0 FPU per gram of cellulose.

Furthermore, the present invention provides the process as describedabove, where, in the step of removing (step iv), at least a portion ofthe hydrolysis product stream is separated from the unhydrolyzed solidsby using a clarifier zone at the top of the unmixed hydrolysis reactor.The hydrolysis product and the unhydrolyzed solids may be removed fromthe clarifier zone at separate locations. Alternatively, at least aportion of the hydrolysis product stream is separated from theunhydrolyzed solids using a solids-liquid separator.

The present invention is directed to the process as described above,wherein, in the step of adding (step iii), the cellulase enzymes arechosen to produce glucose, cellobiose, glucose oligomers, or acombination thereof.

The present invention also provides for the process as described above,wherein, in the step of providing (step i), the pH of the slurry isadjusted from about 4.0 to about 6.0, preferably from about 4.5 to about5.5. Furthermore, the temperature may be from about 45° C. to about 70°C., preferably about 45° C. to about 65° C.

The present invention relates to the process as described above,wherein, in the step of adding (step iii), one or more than oneflocculating compound is used. The flocculating compound may be selectedfrom the group consisting of a cationic polymer, a non-ionic polymer, ananionic polymer, an amphoteric polymer, salts, alum, and a combinationthereof. Preferably, the one or more than one flocculating compound isthe cationic polymer, for example, but not limited to, a polyacrylamide.The flocculating compound may be added at a dosage from about 0.1 toabout 4 kg per tonne solids.

The present invention also relates to the process as described above,wherein the average slurry flow velocity is between about 0.1 and about12 feet per hour, more preferably, between about 0.1 and about 4 feetper hour.

The present invention also provides an enzyme composition comprisingcellulase enzymes and one or more than one flocculent, for hydrolyzingcellulose to glucose, cellobiose, glucose oligomers, or a combinationthereof. Preferably, the cellulase enzymes are produced by Aspergillus,Humicola, Trichoderma, Bacillus, Thermobifida, or a combination thereof.Furthermore, the one or more than one flocculating compound may beselected from the group consisting of a cationic polymer, a non-ionicpolymer, an anionic polymer, an amphoteric polymer, salts, alum, and acombination thereof. Preferably, the one or more than one flocculatingcompound is the cationic polymer, for example a polyacrylamide.

The present invention is also directed to a use of an enzyme compositioncomprising cellulase enzymes and one or more than one flocculent forhydrolyzing cellulose to glucose, cellobiose, glucose oligomers, or acombination thereof.

The present invention is also directed to a use of an enzyme compositioncomprising cellulase enzymes and one or more than one flocculent for theenzymatic hydrolysis of cellulose to produce a hydrolysis product from apre-treated cellulosic feedstock, the use of the enzyme compositioncomprising:

i) providing an aqueous slurry of the pre-treated cellulosic feedstock,the slurry comprising from about 3% to about 30% undissolved solids in aliquid, the undissolved solids comprising at least about 20% cellulose;

ii) introducing the aqueous slurry at the bottom of a hydrolysis reactorand limiting axial dispersion in the reactor by avoiding mixing, andmaintaining an average slurry flow velocity of about 0.1 to about 20feet per hour, such that the undissolved solids flow upward at a rateslower than that of the liquid;

iii) adding the enzyme composition to the aqueous slurry before orduring the step of introducing (step ii); and

iv) removing an aqueous stream comprising hydrolyzed product andunhydrolyzed solids from the hydrolysis reactor, the hydrolysis productcomprising glucose, cellobiose, glucose oligomers, or a combinationthereof.

Preferably, the cellulase enzymes are produced by Aspergillus, Humicola,Trichoderma, Bacillus, Thermobifida, or a combination thereof, and theone or more than one flocculating compound may be selected from thegroup consisting of a cationic polymer, a non-ionic polymer, an anionicpolymer, an amphoteric polymer, salts, alum, and a combination thereof.Preferably, the one or more than one flocculating compound is thecationic polymer, for example, a polyacrylamide.

The present invention also provides an enzyme composition comprisingcellulase enzymes and one or more than one flocculent, for hydrolyzingcellulose to glucose, cellobiose, glucose oligomers, or a combinationthereof, wherein the hydrolysis is carried out by:

i) providing an aqueous slurry of the pre-treated cellulosic feedstock,the slurry comprising from about 3% to about 30% undissolved solids in aliquid, the undissolved solids comprising at least about 20% cellulose;

ii) introducing the aqueous slurry at the bottom of a hydrolysisreactor, limiting axial dispersion in the reactor by avoiding mixing,and maintaining an average slurry flow velocity of about 0.1 to about 20feet per hour, such that the undissolved solids flow upward at a rateslower than that of the liquid;

iii) adding the enzyme composition to the aqueous slurry before orduring the step of introducing (step ii); and

iv) removing an aqueous stream comprising hydrolysis product andunhydrolyzed solids from the hydrolysis reactor, the hydrolysis productcomprising glucose, cellobiose, glucose oligomers, or a combinationthereof.

The present invention provides a kit comprising cellulase enzymes andone or more than one flocculent and instructions for hydrolyzingcellulose to produce a hydrolysis product from a pre-treated cellulosicfeedstock, the instructions comprising:

i) providing an aqueous slurry of the pre-treated cellulosic feedstock,the slurry comprising from about 3% to about 30% undissolved solids in aliquid, the undissolved solids comprising at least about 20% cellulose;

ii) introducing the aqueous slurry at the bottom of a hydrolysis reactorand limiting axial dispersion in the reactor by avoiding mixing, andmaintaining an average slurry flow velocity of about 0.1 to about 20feet per hour, such that the undissolved solids flow upward at a rateslower than that of the liquid;

iii) adding the cellulase enzyme mixture and the one or more than oneflocculent to the aqueous slurry before or during the step ofintroducing (step ii); and

iv) removing an aqueous stream comprising hydrolysis product andunhydrolyzed solids from the hydrolysis reactor, the hydrolysis productcomprising glucose, cellobiose, glucose oligomers, or a combinationthereof.

The present invention also provides a method for preparing an enzymecomposition for use in hydrolyzing cellulose to produce a hydrolysisproduct from a pre-treated cellulosic feedstock, the method comprisingobtaining one or more than one cellulase enzymes from a plant, fungal ormicrobial source, and combining the cellulase enzymes with one or morethan one flocculent to produce the enzyme composition.

The present invention also provides the method for preparing the enzymecomposition as described above, wherein the cellulase enzymes areproduced by Aspergillus, Humicola, Trichoderma, Bacillus andThermobifida.

The present invention provides a system for hydrolyzing cellulose toglucose, cellobiose, glucose oligomers, or a combination thereof, thesystem comprising a feedstock slurry supply line in fluid communicationwith an input to an upflow hydrolysis reactor, a solids-liquid separatorin fluid communication with the upflow hydrolysis reactor and comprisinga first output for withdrawing a slurry comprising unhydrolyzed solidsand a second output for withdrawing a stream comprising hydrolysisproduct, the hydrolysis product comprising glucose, cellobiose, glucoseoligomers, or a combination thereof, wherein the feedstock supply line,the upflow hydrolysis reactor, or both the feedstock supply line and theupflow hydrolysis reactor, comprise an enzyme composition comprisingcellulase enzymes and one or more than one flocculent.

The present invention also provides a system as described above, whereinthe feedstock supply line, when in use, comprises a pre-treatedfeedstock. The solids-liquid separator may be a settling tank, aclarifier, a clarifier zone, a centrifuge or a filter. When the systemis in use, cellulase enzymes may be present at a dosage from about 1.0to about 40.0 FPU per gram of cellulose of the pre-treated feedstock andone or more than one flocculating compound may be present at a dosagefrom about 0.1 to about 4.0 kg per tonne of solids of the pre-treatedfeedstock.

As described herein, the operation of the hydrolysis of cellulose withinan upflow settling reactor may be enhanced by the addition of one ormore flocculating compounds. The flocculating compounds increase thesize of the cellulosic solids, thereby increasing the rate of thesettling of the cellulosic solids. This helps to hold the solids in thereactor for a longer period of time, thereby increasing the degree ofconversion of the cellulose. Furthermore, the process as describedherein provides for the hydrolysis of the feedstock slurry within thehydrolysis reactor in the absence of mixing, in that no active mixing ofthe slurry, through the use of impellers, pumps or the like, within thehydrolysis tank is required.

The use of the upflow settling hydrolysis reactor addresses several ofthe shortcomings of the prior art. The invention improves the efficiencyof the enzymatic hydrolysis of cellulose. This results in a higherdegree of conversion of the cellulose to glucose. Alternatively, theupflow settling reactor results in a lower requirement for cellulaseenzymes than conventional hydrolysis systems. The improved enzymatichydrolysis is achieved without the expense of mixing of the slurrywithin the hydrolysis reactor, and without adding intense shear to thesystem. The improvements associated with the use of an upflow hydrolysisreactor may be enhanced by using a flocculating compound.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features of the invention will become more apparent fromthe following description in which reference is made to the appendeddrawings wherein:

FIG. 1 shows a schematic of a system comprising an upflow reactor thatmay be used in accordance with an embodiment of the present invention.FIG. 1A shows the system comprising an upflow reactor and a settlingtank. FIG. 1B shows the upflow reactor of FIG. 1A. FIG. 1C shows aportion of the system where the upflow reactor comprises a clarifierzone.

FIG. 2 shows the undissolved solids content sampled at various heightswithin the hydrolysis reactor in the absence of adding a flocculent.

FIG. 3 shows the percentage of cellulose conversion measured by samplingat various heights within the hydrolysis reactor in the absence ofadding a flocculent.

FIG. 4 shows the undissolved solids content sampled at various heightswithin the hydrolysis reactor in the presence of a flocculent.

FIG. 5 shows the amount of converted cellulose sampled at variousheights within the hydrolysis reactor in the presence of adding aflocculent.

FIG. 6 shows an increase in efficiency in cellulose conversion in theproduct leaving the hydrolysis reactor in the presence of addedflocculent compared to the absence of flocculent.

DESCRIPTION OF PREFERRED EMBODIMENT

The present invention relates to processes for the conversion ofcellulosic feedstocks into products. More specifically, the presentinvention relates to processes having improved efficiency for enzymaticconversion of cellulosic feedstocks.

The following description is of a preferred embodiment by way of exampleonly and without limitation to the combination of features necessary forcarrying the invention into effect.

The invention relates to a process for the enzymatic conversion ofcellulose to break down products, for example, but not limited to,glucose, cellobiose, glucose oligomers, or a combination thereof. In anaspect of the invention, the process involves pumping an aqueouscellulose slurry with cellulase upward in an unmixed hydrolysis reactor.The upward velocity of the slurry is slow, such that the solidparticles, which are denser than the bulk slurry, tend to flow upwardmore slowly than the liquor. It is well established that cellulaseenzymes bind tightly and preferentially to cellulose. The slow upwardflow of the cellulose-containing solid particles retains thecellulose-containing solids and the bound cellulase enzyme in thereactor for a longer time than the liquid. The retention of celluloseand bound cellulase increases the conversion of cellulose to products,for example, glucose. Near the top of the reactor, the aqueous productor sugar stream and the unhydrolyzed solids are withdrawn. If theproduct is glucose, the aqueous sugar stream is withdrawn forfermentation to ethanol and other further processing. The process asdescribed herein achieves a longer cellulose hydrolysis time within asmaller reactor than would otherwise be required for plug flow of theliquid and solids. Alternatively, the process as described hereinachieves a higher cellulose conversion with less cellulase enzyme thanwould otherwise be required.

The present invention provides a process for the enzymatic hydrolysis ofcellulose to produce a hydrolysis product from a pre-treated cellulosicfeedstock, the process comprising:

i) providing an aqueous slurry of the pre-treated cellulosic feedstock,the slurry comprising from about 3% to about 30% undissolved solids in aliquid, the undissolved solids comprising at least about 20% cellulose;

ii) introducing the aqueous slurry at the bottom of a hydrolysis reactorand limiting axial dispersion in the reactor by avoiding mixing, andmaintaining an average slurry flow velocity of about 0.1 to about 20feet per hour, such that the undissolved solids flow upward at a rateslower than that of the liquid;

iii) adding cellulase enzymes to the aqueous slurry before or during thestep of introducing (step ii); and

iv) removing an aqueous stream comprising hydrolysis product andunhydrolyzed solids from the hydrolysis reactor, the hydrolysis productcomprising glucose, cellobiose, glucose oligomers, or a combinationthereof.

Furthermore, in the step of adding (step iii), a flocculent may also beadded to the slurry, either directly to the slurry, or along with thecellulase enzymes being added to the slurry.

The glucose may then be used for further processing to produce a productof interest, for example, but not limited to, ethanol.

Even though the upflow settling reactor, and process as described hereinare suited to the enzymatic conversion of cellulose to glucose, thisreactor and associated process may also be used to convert cellulose toother products, including, but not limited to, cellobiose (preferably ifthe enzyme, β-glucosidase (βG), is omitted from the cellulase) andglucose oligomers (preferably if the cellobiohydrolase enzymes (CBH) andβG are omitted from the cellulase). To further exemplify the presentinvention, the process for converting cellulose to glucose is described.However, it is to be understood that that this process may be used forthe production of alternate products by incorporating differentcellulase enzyme mixtures during hydrolysis of the feedstock.

By the term “cellulosic feedstock” or “cellulosic material”, it is meantany type of biomass comprising cellulose such as, but not limited to,non-woody plant biomass, agricultural wastes and forestry residues andsugar-processing residues. For example, the cellulosic feedstock caninclude, but is not limited to, grasses, such as switch grass, cordgrass, rye grass, miscanthus, or a combination thereof; sugar-processingresidues such as, but not limited to, sugar cane bagasse and sugar beetpulp; agricultural wastes such as, but not limited to, soybean stover,corn stover, oat straw, rice straw, rice hulls, barley straw, corn cobs,wheat straw, canola straw, oat hulls, and corn fiber; and forestrywastes, such as, but not limited to, recycled wood pulp fiber, sawdust,hardwood, softwood, or any combination thereof. Further, the cellulosicfeedstock may comprise cellulosic waste or forestry waste materials suchas, but not limited to, newsprint, cardboard and the like. Cellulosicfeedstock may comprise one species of fiber or, alternatively,cellulosic feedstock may comprise a mixture of fibers that originatefrom different cellulosic feedstocks. Wheat straw, barley straw, cornstover, soybean stover, canola straw, switch grass, reed canary grass,sugar cane bagasse, cord grass, oat hulls, sugar beet pulp andmiscanthus are particularly advantageous as cellulosic feedstocks due totheir widespread availability and low cost.

In principle, any material that contains a substantial amount ofcellulose is suitable for the process of the present invention. Inpractice, the cellulosic material comprises cellulose in an amountgreater than about 20% (w/w) to produce a significant amount of glucose.The cellulosic material can be of higher cellulose content, for exampleat least about 30% (w/w), 35% (w/w), 40% (w/w) or more. Therefore, thecellulosic material may comprise from about 20% to about 70% (w/w)cellulose, or from 25% to about 70% (w/w) cellulose, or about 35% toabout 70% (w/w) cellulose, or more, or any amount therebetween, forexample, but not limited to, 20, 22, 24, 25, 26, 28, 30, 32, 34, 35, 36,38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68 and 70%(w/w) cellulose.

The present invention may be practiced with a natural cellulosicfeedstock or a cellulosic material that has been processed orpre-treated. Processing and pre-treatment methods are intended todeliver a sufficient combination of mechanical and chemical action so asto disrupt the fiber structure and increase the surface area offeedstock accessible to cellulase enzymes. Mechanical action typicallyincludes, but is not limited to, the use of pressure, grinding, milling,agitation, shredding, compression/expansion, or other types ofmechanical action. Chemical action can include, but is not limited to,the use of heat (often steam), acid, and solvents. Several chemical andmechanical pre-treatment methods are well known in the art.

One approach to pre-treatment of the feedstock is steam explosion, usingthe process conditions described in U.S. Pat. No. 4,461,648, and U.S.Pat. No. 4,237,226 (which are herein incorporated by reference). In thisprocess, lignocellulosic biomass is loaded into a steam gun in thepresence of 0% to 5% (v/v), or any amount therebetween, sulfuric acid orany other suitable acid. The steam gun is then filled rapidly with steamto a temperature of about 160° C. to about 280° C., or any amounttherebetween, and held at high pressure for a cooking time of betweenabout 3 seconds to about 30 minutes, or any amount therebetween. Thevessel is then rapidly depressurized to expel the pre-treated biomass.Any parameters known in the prior art to effect steam explosionpre-treatments such as, but not limited to, those described in Foody, etal., (Final Report, Optimization of Steam Explosion Pre-treatment, U.S.DEPARTMENT OF ENERGY REPORT ET230501, April 1980; which is hereinincorporated by reference) may be used in the method of the presentinvention. The conditions chosen for steam explosion will depend uponthe nature of the feedstock and the desired degree of susceptibility toenzymes. However, other methods that are known within the art may beused as required for preparation of a pre-treated feedstock, forexample, but not limited to, those disclosed in U.S. Pat. No. 5,846,787(Ladisch), U.S. Pat. No. 5,198,074 (Villavicencio), U.S. Pat. No.4,857,145 (Villavicencio), or U.S. Pat. No. 4,556,430 (Converse; whichare incorporated herein by reference), ammonia freeze explosion (U.S.Pat. No. 5,171,592, Holtzapple) and concentrated alkali treatment.

Regardless of whether a pre-treatment step is performed, the cellulosicfeedstock may optionally be washed with water, or leached with water,for example, as disclosed in WO 02/070753 (Griffin et al., which isincorporated herein by reference) prior to enzymatic hydrolysis. Washingof pre-treated cellulosic feedstock can remove inhibitors of cellulaseenzymes such as dissolved sugars and sugar degradation products,dissolved lignin and phenolic compounds, and other organic compounds inthe system. The concentration of cellulose within washed pre-treatedfeedstock typically increases, for example up to levels of about50%-70%.

The cellulosic material is slurried in a liquid at a concentration thatis thick and can still be pumped. For example, but without wishing to belimiting, the liquid may be water, a recycled process stream or treatedeffluent. The concentration of cellulosic feedstock in the slurrydepends on the material, but may be between about 3% to about 30% (w/w)undissolved solids, or any concentration therebetween, for example, fromabout 5% to about 20%, or from about 10% to about 20% undissolvedsolids, or any amount therebetween. For example, the concentration ofcellulosic feedstock in the slurry may be 3, 5, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28 or 30% undissolved solids (w/w). As is well knownin the art, the concentration of suspended or undissolved solids can bedetermined by filtering a sample of the slurry using glass microfiberfilter paper, washing the filter cake with water, and drying the cakeovernight at 105° C.

The pH of the slurry is generally adjusted to within the range ofoptimum pH for the cellulase enzymes used. Generally, the pH of theslurry is adjusted to within the range of about 3.0 to about 7.0, orabout 4.0 to about 6.0, or any pH therebetween, preferably within therange of about 4.5 to about 5.5. For example, the pH may be about 3.0,3.5, 4.0, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 6.0,6.5 or 7.0. The pH of the slurry may be adjusted using any suitable acidor base known in the art. For example, sodium hydroxide, ammonia,ammonium hydroxide, potassium hydroxide or other suitable base (if theslurry is acidic), or sulfuric acid, or other suitable acid (if theslurry is alkaline), may be used. However, the pH of the slurry can behigher or lower than about 4.5 to 5.5 if the cellulase enzymes used arealkalophilic or acidophilic, respectively. The pH of the slurry shouldbe adjusted to within the range of optimum pH for the enzymes used.

The temperature of the slurry is adjusted to the point that is withinthe optimum range for the activity of the cellulase enzymes. Generally,a temperature of about 45° C. to about 70° C., or about 45° C. to about65° C., or any temperature therebetween, is suitable for most cellulaseenzymes. For example, the temperature of the slurry may be adjusted toabout 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69 or 70° C. However, the temperature ofthe slurry may be higher for thermophilic cellulase enzymes.

Cellulase enzymes are then added to the slurry. By the term “cellulaseenzymes”, “cellulase”, or “enzymes”, it is meant enzymes that catalysethe hydrolysis of cellulose to products such as glucose, cellobiose, andother cellooligosaccharides. Cellulase is a generic term denoting amultienzyme mixture comprising exo-cellobiohydrolases (CBH),endoglucanases (EG) and β-glucosidases (βG) that can be produced by anumber of plants and microorganisms. The process of the presentinvention can be carried out with any type of cellulase enzymes,regardless of their source; however, microbial cellulases are generallyavailable at lower cost than those of plants. Among the most widelystudied, characterized, and commercially produced cellulases are thoseobtained from fungi of the genera Aspergillus, Humicola, andTrichoderma, and from the bacteria of the genera Bacillus andThermobifida. Cellulase produced by the filamentous fungi Trichodermalongibrachiatum comprises at least two cellobiohydrolase enzymes termedCBHI and CBHII and at least 4 EG enzymes.

Cellulase enzymes work synergistically to degrade cellulose to glucose.CBHI and CBHII generally act on the ends of the glucose polymers incellulose microfibrils liberating cellobiose (Teleman et al. 1995,European J. Biochem 231:250-258), while the endoglucanases act at randomlocations on the cellulose. Together these enzymes hydrolyse celluloseto smaller cello-oligosaccharides such as cellobiose. Cellobiose ishydrolysed to glucose by β-glucosidase.

The cellulase enzyme dosage added to the slurry is chosen to achieve asufficiently high level of cellulose conversion without overdosing. Forexample, an appropriate cellulase dosage can be about 1.0 to about 40.0FPU per gram of cellulose, or any amount therebetween. For example, thecellulase dosage may be about 1.0, 3.0, 5.0, 8.0, 10.0, 12.0, 15.0,18.0, 20.0, 22.0, 25.0, 28.0, 30.0, 32.0, 35.0, 38.0 or 40.0 FPU pergram, or any amount therebetween. The FPU (Filter Paper Unit) is astandard measurement familiar to those skilled in the art and is definedand measured according to Ghose (1987, Pure and Appl. Chem. 59:257-268).For complete conversion to glucose, it is preferred that the cellulasecontain an adequate quantity of β-glucosidase (cellobiase) activity. Thedosage level of β-glucosidase is about 5 to about 600 β-glucosidaseunits per gram of cellulose, or any amount therebetween. A typicaldosage level of β-glucosidase is about 10 to about 400 β-glucosidaseunits per gram of cellulose, or any amount therebetween; for example,the dosage may be 10, 12, 15, 17, 20, 22, 25, 27, 30, 32, 35, 37, 40,42, 45, 47, 50, 52, 55, 57, 60, 62, 65, 67, 70, 72, 75, 77, 80, 82, 85,87, 90, 92, 95, 97, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280,300, 320, 340, 360, 380 and 400 β-glucosidase units per gram ofcellulose, or any amount therebetween. The β-glucosidase unit ismeasured according to the method of Ghose (1987, Pure and Appl. Chem.59:257-268).

The cellulase enzymes may be handled in an aqueous solution, as a powderor as a granulate. The enzymes may be added to the slurry at any pointprior to its entry into the reaction vessel (also referred to as ahydrolysis tower or hydrolysis reactor; 110 or 110′, FIG. 1). Forexample, but without wishing to be limiting, the cellulase enzymes maybe added to the slurry immediately prior to entering the hydrolysistower. The enzymes may be mixed into the slurry using mixing equipmentthat is familiar to those of skill in the art. In a non-limitingexample, a small make-up tank (90, FIG. 1A) located upstream of the mainhydrolysis reactor (110 or 110′) may be used for adding the enzymes tothe slurry, adjusting the pH and achieving the desired temperature ofthe slurry.

With reference to FIG. 1A, the feedstock 10 is pre-treated as describedabove. This stream is cooled using a heat exchanger 20 that exchangesagainst product stream 30 or other suitable fluid. The slurry 40 maythen be further cooled using a second fluid, for example cold water 45,at heat exchanger 50. The slurry may then be pumped into a hydrolysismake-up tank 90, along with cellulase enzymes 70 and ammonium hydroxide80, to adjust the pH. In this example, the contents of the hydrolysismake-up tank 90 are mixed and pumped out of the make-up tank 90, alongpipe 100, to the hydrolysis tank 110. However, the cellulase enzymes maybe mixed with the feedstock elsewhere, for example, at 190, 195 or 197or within a line that feeds the hydrolysis reactor, including, but notlimited to, line 10, 40 or 100, or a combination thereof.

By the term “hydrolysis tower”, “upflow hydrolysis reactor”, “hydrolysisreactor” “hydrolysis tank”, or “upflow settling reactor”, it is meant areaction vessel (tower) of appropriate construction to accommodate thehydrolysis of cellulosic slurry by cellulase enzymes, for example 110(FIG. 1B) or 110′ (FIG. 1C). The hydrolysis tank may be jacketed withinsulation, steam, hot water, electrical heat tracing, or other heatsource to maintain the desired temperature. In the present application,the hydrolysis reactor is an unmixed hydrolysis reactor, in the sensethat no mixing of the reactor contents takes place during the hydrolysisreaction. As set out below, some small amount of localized mixing of thereactor contents may occur due to the small amount of power inputassociated with the addition and withdrawal of solids and liquids fromthe system. The slurry and cellulase mixture may enter the upflowsettling reactor directly at the bottom and be pumped upward in thehydrolysis tower; alternatively, the slurry can be pumped downwardthrough a pipe located in the centre (e.g. 105) of the reactor andemerge at the bottom of the reactor to flow upward, surrounding thepipe. The latter configuration is advantageous in that heat from theslurry can be captured in the hydrolysis reactor. Once the slurryreaches the bottom of the hydrolysis tank 110 or 110′, the slurry movesupward and is dispersed across the width of the hydrolysis tank; axialdispersion (i.e. dispersion along the height of the tank) is minimizedby avoiding mixing. The slurry flow velocity is chosen such that theliquid component of the slurry flows upward at a rate faster than thatof the undissolved solids. The hydrolysis tower is designed such thatthe contents of the slurry are relatively uniform in the radialdirection, at any given height. The uniform distribution may be achievedusing distributors (e.g. 120), or other equipment well known in the art.This may include fractal distributors (for example, Rohn Haas AdvancedAmerpack™ system manufactured by Amalgamated Research Inc.) or arotating wand. For example, in FIG. 1B, a vertical control shaftsupports a pair of cantilevered truss arms at the bottom of the reactor110 and another pair at the top of the reactor. The arms incorporate aheader system with nozzles to distribute the product at the bottom ofthe vessel and to collect it at the top. A two-port rotary joint is usedfor the feed and the discharge from the central shaft. The distributionof the slurry within the hydrolysis tank is achieved in the absence ofactive mixing by impellers or pumps.

FIG. 1C shows an alternate hydrolysis tower 110′ comprising a clarifierzone 135 positioned at the top of the tank. As described in more detailbelow, the majority of the solids are withdrawn at the top of zone 130.Excess clarified liquid continues to flow upward in zone 135 and iswithdrawn as clear liquid.

The hydrolysis reactor 110 or 110′ may be of any dimensions that willmaintain a relatively uniform slurry concentration across the reactor,as described. Without wishing to be limiting, the hydrolysis tower mayhave a diameter of between about 10 feet to about 130 feet (3 to 40 m),or any amount therebetween; for example, the diameter of the reactor maybe 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,95, 100, 105, 110, 115, 120, 125, or 130 feet, or any amounttherebetween. The height of the hydrolysis reactor can be of any height,provided that the reactor achieves the purposes herein described.Without wishing to be limiting, the reactor height may be of about 5 toabout 75 feet or about 5 to about 65 feet (1.5 to 23 m), or any amounttherebetween, preferably from about 20 to about 65 feet; for example,the reactor height may be about 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5,25, 27.5, 30, 32.5, 35, 37.5, 40, 42.5, 45, 47.5, 50, 52.5, 55, 57.5,60, 62.5, or 65 feet. The height-to-diameter ratio of the hydrolysisreactor may be between about 0.5 to about 10, or any ratio therebetween;preferably the height-to-diameter ratio is from about 0.5 to about 3.The overall size of the reactor should be chosen so as to avoid placingan undue burden on the foundation supporting the reactor when it isfilled with water. In a non-limiting example, a hydrolysis reactorhaving a diameter of 110 feet and height of 65 feet would have a volumeof 4.60 million gallons.

The slurry is pumped upward into the reactor 110 or 110′ at an averageslurry flow velocity that allows the liquid to flow uniformly up thereactor while the cellulose-containing particles, which are denser thanthe liquid, flow up the reactor more slowly than the liquid, settle, andpack to some solids concentration that is higher than the feed solidsconcentration. The “average flow velocity” of the slurry or “slurry flowvelocity” is the height of the hydrolysis reactor divided by the nominalslurry residence time, based on the reactor volume and the sluiry flowrate to the reactor. For example, a slurry feed rate of 10,000 gallonsper hour to a 120,000 gallon hydrolysis reactor that is 30 feet tall hasa nominal slurry residence time of 120,000 gallons/(10,000gallons/hr)=12 hours and an average slurry flow velocity of 30 ft/12hr=2.5 ft/hr. The average flow velocity is selected to permit solids toflow upward at a slower rate than the average slurry flow velocity. Thispermits the solids to have a longer average residence time in thehydrolysis reactor than the nominal slurry residence time. This differsfrom slurry plug flow reactors in which the flow velocity and theretention time of the liquid and solids in the reactor are substantiallythe same. The flow velocity at which this is achieved will be dependenton the feedstock and the size of the solid particles in the slurry, aswell as the presence of any added flocculent. The use of a flocculentmay permit the use of a higher average flow velocity. Generally, theaverage flow velocity is about 0.1 to about 20 feet per hour, or anyvelocity therebetween. Preferably, the average flow velocity is betweenabout 0.1 to about 12 feet per hour, or any amount therebetween. Morepreferably, the average flow velocity is between about 0.1 to about 4.0feet per hour, or any amount therebetween. For example, the averageslurry flow velocity may be about 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 1.5,2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5,9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0 12.5, 13.0, 13.5, 14.0, 14.5,15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, or 20.0 feetper hour. The nominal slurry residence time in the hydrolysis reactor istypically 4-120 hours, preferably 12-100 hours and most preferably20-100 hours.

Using the method of the present invention, the properties of the slurrywill change as hydrolysis of cellulose proceeds. Without wishing to bebound by theory, during the hydrolysis reaction, the cellulase enzymesbind to the cellulose and therefore remain bound to thecellulose-containing solid particles in the slurry. The average upwardvelocity of the slurry is slow, such that the solid particles, which aredenser than the bulk slurry, tend to flow upward more slowly than theliquor. The slow upward flow of the cellulose-containing solid particlesretains the cellulose-containing solids and the bound cellulase enzymein the reactor for a longer time than the liquid. As the bound enzymesdigest the cellulose and release glucose into solution, the amount ofcellulose, and the density of the solid particles, changes. Depending onthe altered density, smaller particles will flow upward with the liquidor settle to the bottom of the reactor. Due to the differentialretention of the cellulose-containing particles relative to the liquor,the concentration of cellulose will decrease from the bottom to the topof the hydrolysis reactor while the concentration of glucose willincrease from the bottom to the top of reactor. The decrease in theconcentration of cellulose, and an increase in the concentration ofglucose, occurs in the “hydrolysis zone” 130 of the hydrolysis reactor110 (see FIGS. 1A and 1B). The aqueous sugar stream, the unhydrolyzedsolids, and any nearby cellulose-containing particles are withdrawn 150near the top of the hydrolysis zone 140 of the reactor 110. At least aportion of the solids are then separated from the glucose stream, forexample using a solids-liquid separator, for example, settling tank 160and the product stream 30 is sent for fermentation to ethanol and otherfurther processing (170). The longer retention of cellulose within thehydrolysis tower increases the extent of conversion of the cellulose toglucose, thereby achieving a longer cellulose hydrolysis time with asmaller reactor than would be achieved with a mixed reactor.Alternatively, a higher cellulose conversion is achieved with a lowerenzyme dosage than would be required otherwise.

By the term “unhydrolyzed solids” or “unconverted solids”, it is meantcellulose that is not digested by the cellulase enzyme, as well asnon-cellulosic, or other, materials that are inert to cellulase, presentin the feedstock. For example, but without wishing to be limiting in anymanner, the unconverted solids may comprise lignin, silica or othersolid material. As the cellulose in the feedstock is hydrolyzed, theconcentration of unconverted solids within the cellulose-containingsolid particles increases. Depending on the density and particle size,the unconverted solids may be removed with the products at 150 or settleto the bottom in a sediment or sludge 180. If a sludge layer forms atthe bottom of the reactor due to very heavy particles, any means knownin the art may be employed to remove the sludge or sediment. In anon-limiting example, a scraper may be used to remove the sludge. In afurther example, the bottom of the reactor may be tapered to provide apath in which the heaviest solids may settle, and be removed (e.g. line167) and sent for lignin processing 165.

The aqueous glucose, unconverted solids and other particles that arefound near the top 140 of the hydrolysis reactor 110 can be removed as astream 150. Following withdrawal from the top of the reactor, at least aportion of the unconverted solids may be separated from the solublesugar stream. Removal of the unconverted solids can be accomplishedusing a solids-liquid separator, for example by filtration (for example,a filter press, belt filter, drum filter, vacuum filter or membranefilter), centrifugation, settling, for example, settling tank 160, aninclined settler (for example, as disclosed in Knutsen and Davis, 2002,Appl., Biochem. Biotech., 98-100:1161-1172 and Mores et al., 2001, Appl.Biochem. Biotech., 91-93:297-309, both of which are incorporated hereinby reference), a clarifier, or any other suitable process as would beknown in the art. The clarifier may comprise a number of inclined platesto facilitate the separation of the solids and liquid or other featuresthat are known in the art of solids-liquid separation. The solubleglucose, essentially free of undissolved solids, is then suitable forfermentation to ethanol (170). The unconverted solids are primarilylignin, which can be burned and used as fuel for the plant.

Alternatively, the aqueous glucose stream is withdrawn at a locationseparate from the withdrawal of unconverted solids. An alternativemethod for separating the unconverted solids from the glucose is to usethe reactor 110′ in FIG. 1C with a hydrolysis zone 130 extending fromthe bottom of the hydrolysis tower to a level about 65% to about 85% ofthe way up, and a “clarifier zone” 135 directly above the hydrolysiszone. The hydrolysis stream is pumped from the top portion of thehydrolysis zone 130 into the clarifier zone 135. A majority of thesolids are removed at the top of the hydrolysis zone 130. For example,but not wishing to be limiting in any manner, a horizontal wand withnozzles is passed back and forth across the top of the reactor at timeintervals, and the solids-rich stream is withdrawn into the wand andpumped out of the reactor (162). Excess clarified liquid continues toflow upward into clarifier zone 135. In the clarifier zone 135, thesolids generally settle to the level of the wand, while the aqueoussugar stream, essentially free of solids, is removed (30) from the top.The clarifier zone may comprise a number of inclined plates tofacilitate the separation of the solids and liquid and other featuresthat are known in the art of solids-liquid separation. The unconvertedsolids (or unhydrolyzed solids) may be transferred to a solids-liquidseparator to separate at least a portion of the hydrolysis product fromthe unhydrolyzed solids.

If so desired, the cellulose-containing solids obtained by separationfrom the glucose stream can be recycled back into the upflow settlingreactor, or the hydrolysis zone, with the incoming feedstock for furtherconversion to glucose.

It should be appreciated that some small amount of localized mixing ofthe reactor contents may occur due to the small amount of power inputassociated with the addition and withdrawal of solids and liquids fromthe system. For example, localized mixing may occur due to the action ofthe distributors 120, wands or pump(s) that feed the slurry into thehydrolysis reactor. For best operation, the power required for carryingout addition and withdrawal of solids and liquids associated with theoperation of the hydrolysis reactor does not exceed 0.1 HP/1000 gal. Thepower associated with these upflow reactor functions may be between0.001 and 0.1 HP/1000 gal, or any range therebetween. For example, thepower associated with these upflow reactor functions may be 0.001,0.003, 0.008, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09 or0.1 HP/1000 gal. There are no impellers, agitators, eductors, or otherequipment in the reactor specifically designed to mix the slurry.

If more than one hydrolysis reactor is employed, the reactors may be runin a series of two or more than two reactors, in which case the outletof a first reactor feeds the inlet of a second reactor. Alternatively,the reactors may be run in parallel. Furthermore, some of the reactorsin the sequence may be run in series, while others may be run inparallel.

It should also be appreciated that one or more other reactor types inaddition to the upflow reactor may be utilized, such as one or morebatch or continuous stirred reactors. In a non-limiting example, theoutlet of a continuous stirred reactor feeds the inlet of an upflowreactor. As would be apparent to one of skill in the art, othercombinations of reactor types may be used in the present invention.

In an alternative aspect of the present invention, flocculatingcompounds may be added to the slurry to enhance the efficiency of thepresent invention. Flocculating compounds are typically polymers thatare cationic, nonionic, anionic, or amphoteric (containing a mixture ofcharged groups), or salts such as alum. Without wishing to be bound bytheory, flocculating compounds serve to aggregate the solids within thehydrolysis reactor to ensure a more complete exposure to the enzymemixture.

In practising the present invention, one flocculating compound, or amixture containing more than one flocculating compound, may be used. Theflocculent may be provided in any suitable form for addition to theslurry; for example, the flocculent may be a powder, a liquid, or adispersion; for example, the dispersion may be a flocculent slurried inoil or an aqueous solution. A non-limiting example of a suitableflocculent is a cationic polymer, more specifically, a polyacrylamide.Such flocculants include, but are not limited to, CA4500 (SNF Floerger®,France) and Zetag® 7651 (Ciba® Specialty Chemicals, Canada).

The amount of flocculent used will be determined by the amount necessaryto aggregate the solids in the upflow reactor. A person of skill in theart will be able to determine the amount of flocculent to add that wouldaid in solids-aggregation, without the addition of undesirable cost tothe overall process. For example, but without wishing to be limiting,the amount of flocculent added may be an amount in the range of about0.1 to about 4.0 kg per tonne solids or any amount therebetween, orabout 0.5 to about 2.0 kg per tonne solids, or any amount therebetween.For example, the amount of flocculent added may be about 0.1, 0.5, 0.75,1.0, 1.25, 1.5, 1.75, 2.0, 2.5, 3.0, 3.5, or 4.0 kg per tonne solids.

Flocculating compounds may be added directly to the slurry, or dispersedand diluted in water prior to addition to the slurry. The flocculatingcompounds can also be mixed with the cellulase enzymes before additionto the slurry. Dispersion of the flocculent may help ensure uniformapplication of the flocculent in the system. In a non-limiting exampleof the present invention, the flocculent may be dispersed in water at aconcentration of about 0.01% to about 25% by weight, or any amounttherebetween; for example, the concentration of flocculent may be about0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4,0.45, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 7.0, 10.0, 12.0, 14.0, 16.0, 18.0,20.0, 22.0, or 25.0% by weight. The flocculent may be dispersed at thisconcentration by mixing for an appropriate amount of time, for exampleabout 1 minute to about 1 hour. The dispersed flocculent may then beadded directly to the cellulose slurry, or be further diluted in waterto a concentration of about 0.01% to about 1.0%, by weight, or anyamount therebetween, prior to addition to the hydrolysis reactor. Forexample, the concentration of the further diluted flocculent may be0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0% by weight.

The present invention therefore provides a system for hydrolyzingcellulose to glucose, cellobiose, glucose oligomers, or a combinationthereof, the system comprising a feedstock slurry supply line (e.g. 10,40, 100) in fluid communication with an input to an upflow hydrolysisreactor (e.g. 110 or 110′), a solids-liquid separator (e.g. 160 or 135)in fluid communication with the upflow hydrolysis reactor and comprisinga first output for withdrawing a slurry comprising unhydrolyzed solidsand a second output for withdrawing a stream comprising hydrolysisproduct, the hydrolysis product comprising glucose, cellobiose, glucoseoligomers, or a combination thereof, wherein the feedstock supply line,the upflow hydrolysis reactor, or both the feedstock supply line and theupflow hydrolysis reactor, comprise an enzyme composition comprisingcellulase enzymes and one or more than one flocculent. Preferably, thefeedstock supply line comprises a pre-treated feedstock prepared asoutlined above. Furthermore, the cellulase enzymes within the system arepresent at a dosage from about 1.0 to about 40.0 FPU per gram ofcellulose of the pre-treated feedstock, and the one or more than oneflocculating compound is present at a dosage from about 0.1 to about 4.0kg per tonne solids of the pre-treated feedstock.

The present invention also pertains to a use of an enzyme compositioncomprising cellulase enzymes and one or more than one flocculent, forhydrolyzing cellulose to glucose, cellobiose, glucose oligomers, or acombination thereof, as described herein. As this system results inabout 60 to about 98% conversion of the cellulose to glucose, theglucose may be used for the production of ethanol. Preferably, thecellulase enzymes are produced by Aspergillus, Humicola, Trichoderma,Bacillus, Thermobifida, or a combination thereof. The one or more thanone flocculating compound may be selected from the group consisting of acationic polymer, a non-ionic polymer, an anionic polymer, an amphotericpolymer, salts, alum, and a combination thereof. Preferably, the one ormore than one flocculating compound is the cationic polymer, for examplea polyacrylamide. The cellulase enzymes and one or more than oneflocculent as described herein may also be used for the preparation ofan enzyme composition for hydrolyzing cellulose to glucose, cellobiose,glucose oligomers, or a combination thereof. Therefore, the presentinvention also provides a use of an enzyme composition comprisingcellulase enzymes, and one or more than one flocculent, for hydrolyzingcellulose to glucose, cellobiose, glucose oligomers, or a combinationthereof, for the production of ethanol.

The flocculent, or the diluted flocculent may be added prior to thepoint of enzyme addition (195; FIG. 1A) to the slurry, at the point ofenzyme addition (190), after the point of enzyme addition (197), or acombination of these locations. Furthermore, the flocculent may be addedto the slurry at or near the bottom of the hydrolysis reactor (192;FIGS. 1A, 1B and 1C), in the midsection of the hydrolysis reactor (191),at the top of the reactor (193), at a location outside the reactor (195,197), or a combination of these locations. In a non-limiting example,flocculent may be added at the bottom (192) and in the midsection (191)of the reactor. If the flocculent inhibits or negatively impacts onenzyme activity, then the flocculent should not be in direct contactwith the enzyme in a manner that might be deleterious to the enzymeprior to addition to the slurry or hydrolysis tank.

The flocculent may be added by pumping through a series of valves andelbows. Such a configuration may improve mixing of the flocculent withthe slurry and prevent backing up of the mixture into the system. In analternate example, an in-line mixer can be used to impart turbulence tothe flocculent and thereby enhance the dispersion. Alternatively, theflocculent may be added to the slurry (e.g. at 190) in the make-up tank90, then pumped to the hydrolysis reactor 110 or 110′ along with theslurry and cellulase enzymes 40.

Once in the hydrolysis reactor 110 or 110′, the flocculent proceeds tobind to the solid particles and aid in the aggregation in the hydrolysiszone 130. Without wishing to be bound by theory, this helps prevent thecellulose-containing particles from being removed out the top of thereactor, from settling in the sludge layer by keeping them suspended inthe reactor, or a combination thereof. Binding the solid particlesallows greater residence time for the cellulose-containing particleswithin the hydrolysis zone (130) and results in a more complete andefficient digestion of cellulose by the enzymes. At the top of theupflow reactor 140 the flocculent is primarily bound to the unconvertedsolids, and exits the reactor (150; FIG. 1A) with the particles.

It is contemplated that glucose produced by the hydrolysis of cellulosefrom the pre-treated feedstock that leaves the reactor 110 or 110′through line 30 may be fermented to ethanol (170). Fermentation ofglucose and other sugars to ethanol may be performed by conventionalprocesses known to those skilled in the art, and may be effected by avariety of microorganisms including yeast and bacteria or geneticallymodified microorganisms, for example, but not limited those described inWO 95/13362, WO 97/42307, or Alcohol production from Cellulosic Biomass:The Iogen Process (in: The Alcohol Textbook, Nottingham UniversityPress, 2000; which are herein incorporated by reference). Ethanolproduction and recovery are performed using well-established processesknown to one of skill in the art in the alcohol industry.

As indicated previously, the upflow settling reactor is suited to theenzymatic conversion of cellulose to glucose. However, this type ofsystem can be used to convert cellulose to other products, includingcellobiose (preferably if βG is omitted from the cellulase) and glucoseoligomers (preferably if CBH and βG are omitted from the cellulase).

The method of the present invention increases the length of time thatthe solids are in the reactor, which increases the contact time betweenthe cellulase enzymes and cellulose because the enzymes remain bound tothe cellulose. This in turn increases the efficiency of the hydrolysisprocess by increasing the degree of cellulose conversion obtained in ahydrolysis reactor of a given size. Thus, the costs of enzymatichydrolysis are minimized. Furthermore, the upflow settling method doesnot require agitation within the reactor, saving the power and equipmentcosts associated with mixing.

The above description is not intended to limit the claimed invention inany manner. Furthermore, the discussed combination of features might notbe absolutely necessary for the inventive solution. The presentinvention will be further illustrated in the following examples.However, it is to be understood that these examples are for illustrativepurpose only, and should not be used to limit the scope of the presentinvention in any manner.

EXAMPLES Example 1 Large Scale Upflow Hydrolysis Reactor

Pre-treated wheat straw is prepared using the method of U.S. Pat. No.4,461,648 (Foody, which is incorporated herein by reference). Thepre-treated material is an aqueous slurry of 7.8% undissolved solids, ata temperature of 70° C., and a mass flow rate of 553 t/hr. This aqueousslurry is cooled to 60° C. in a heat exchanger (20), against the productstream (30). The 60° C. slurry is then cooled to a final temperature of50° C. by cold water at heat exchanger (45). The slurry is pumped intothe hydrolysis make-up tank (90; volume 86,000 gallons) along withcellulase enzymes (70; 5 FPU/g cellulose) and ammonia (80; 1200 gramsper tonne wet slurry), to adjust the pH to 4.5 to 5.0. The contents ofthe hydrolysis make-up tank are mixed for a residence time of 40 minutesand then the combined stream (100) is pumped out of the make-up tank anddown a pipe through the middle (105) of the hydrolysis tank (110). Thehydrolysis tank is jacketed with 15 psig steam used to maintain 50° C.At the bottom of the hydrolysis tank, the slurry is pumped upward anddispersed (120) across the width of the hydrolysis tank.

The hydrolysis tank has a volume of 4.6 million gallons. The liquidflows upward in the hydrolysis tank faster than the solids, which settleto a concentration of about 10% throughout the tank. The cellulose isheld up in the tank 188 hrs, which is long enough to convert 92% of thecellulose to glucose. The aqueous sugar stream and the unhydrolyzedsolids (150) flow out of the top of the tank and are pumped to thesettling tank (160). In the settling tank, which had a volume of 1.12million gallons, the solids settle to the bottom to a concentration of10% and are pumped out via line 162 to a lignin filter press 165 torecover sugar from the solids by pressing and washing. The sugar fromthis stream is combined with the sugar stream from the top of thesettler, which is then sent for fermentation to ethanol (170).

Example 2 Upflow Hydrolysis with Trichoderma Cellulase Enzyme

Wheat straw was pre-treated using the method of U.S. Pat. No. 4,461,648(Foody, which is incorporated herein by reference). The pre-treatedmaterial was slurried in water at a concentration of 3.7% undissolvedsolids and the pH was adjusted to 5.5 with 30% sodium hydroxide. Theundissolved solids were 55% cellulose. The slurry was pumped at a rateof 40 liters per minute into the bottom of a vertical hydrolysis reactor(110; FIG. 1C). This corresponds to an upward flow velocity of 0.7feet/hr. The tower volume was 144,700 liters, of which the hydrolysiszone (130) was the lower 115,000 liters (a height of 34.4 feet) and thetop 29,700 liters was a clarifier (135). The diameter of the tower was3.8 meters and the height was 13.5 meters (44.3 feet). The temperatureof the slurry was 55° C. upon entry in the reactor, and graduallydecreased to 50° C. near the top of the reactor. The cellulase enzyme,obtained from Trichoderma (available from Iogen Bioproducts, Ottawa) wasadded to the slurry in the hydrolysis make up tank (90; FIG. 1A) at adosage of 36 FPU per gram cellulose, added to the line before enteringthe tower as in Example 1.

The slurry containing pre-treated solids and cellulase was pumped intothe bottom of the tower 105 and hydrolysis took place as the slurryflowed up the tower. At the top of the hydrolysis zone, which was 79% ofthe volume of the tower, the slurry was transferred to a clarifier zone135. A stream containing settled solids was withdrawn (162; FIG. 1C) atthe interface of the hydrolysis zone and the clarifier zone. A secondstream 30 containing glucose in the aqueous phase with littleundissolved solids was collected at the top of the clarifier zone.

The solids profile in the reactor over the course of the run is shown inFIG. 2. At a level of 1 foot above the bottom, the undissolved solidssettled to a concentration of 8% to 14%, by weight. This wassignificantly more concentrated than the feed concentration of 3.7%undissolved solids. At points higher than this, the solids concentrationwas lower.

The cellulose conversion profile is shown in FIG. 3. The degree ofcellulose conversion was 73% to 83% early in the run, and by the end was95% at the heights of 15 feet and 24 feet. The glucose concentration inthe stream flowing out of the upflow reactor was 25 g/L. This representsa good level of cellulose conversion and glucose production obtainedwithout mixing a reactor, providing shear, or otherwise moving thematerial beyond pumping it slowly up the tower.

Example 3 Upflow Hydrolysis with Trichoderma Cellulase Enzyme inPresence of Flocculating Compound

A hydrolysis of pre-treated wheat straw was carried out as described inExample 2 with 4.4% undissolved solids, except that a flocculent wasadded to improve the settling of the solids. A cationic polymer, CA4500(SNF Floerger®, France), was added at a dosage of 2 kg per tonneundissolved solids and dispersed inline upon addition after the point ofenzyme addition to the slurry.

The solids profile in the reactor over the course of the run is shown inFIG. 4. At a level of 1 foot above the bottom, the undissolved solidssettled to a concentration of 6% to 10%, by weight, similar to thatobserved in Example 2, and was significantly more concentrated than thefeed concentration of 4.4% undissolved solids. At points higher thanthis within the reactor, the solids concentration was 5.5% to 8%. Thisindicated the flocculent was effective at aggregating and settling thesolids.

The cellulose conversion profile is shown in FIG. 5. The degree ofcellulose conversion was 65% to 85% early in the run, and by the end was85% to 92% at the heights of 15 feet and 24 feet. The glucoseconcentration in the stream flowing out of the reactor was 27 g/L. Thisrepresents a good level of cellulose conversion obtained without mixinga reactor, providing shear, or otherwise moving the material beyondpumping it slowly up the tower.

Example 4 Cellulose Conversion at Various Enzyme Levels in Presence andAbsence of Flocculating Compound

The hydrolysis reactions described in Examples 2 and 3 showed a similarfinal glucose concentration, which may be due to the presence of anexcess of enzymes. In order to determine the effect of flocculent onhydrolysis efficiency, hydrolysis was performed in the presence andabsence of flocculent at various enzyme dosages.

A hydrolysis of pre-treated wheat straw is carried out as described inExamples 2 and 3, except that the amount of cellulase enzyme added isvaried. A hydrolysis with 8, 12, 16, 20, 24, 28, 32, and 36 FPU enzymeis carried out in the presence and absence of flocculent for 48 hours.The cellulose conversion (in %) is measured and shown in FIG. 6.

As shown in FIG. 6, at lower enzyme dosages, the use of flocculentresults in increased cellulose conversion. This represents an overallsaving in the cost of cellulose conversion.

1. (canceled)
 2. The system according to claim 58, wherein the feedstockslurry is introduced at the bottom of the hydrolysis reactor with auniform radial distribution. 3-47. (canceled)
 48. The system accordingto claim 58, wherein the average slurry flow velocity is between about0.1 and about 12 feet per hour.
 49. The system according to claim 48,wherein the average slurry flow velocity is between about 0.1 and about4 feet per hour. 50-57. (canceled)
 58. A system for hydrolyzingcellulose to glucose, cellobiose, glucose oligomers, or a combinationthereof, the system comprising a feedstock slurry supply line in fluidcommunication with an input to an upflow hydrolysis reactor, asolids-liquid separator in fluid communication with the upflowhydrolysis reactor and comprising a first output for withdrawing aslurry comprising unhydrolyzed solids and a second output forwithdrawing a stream comprising hydrolysis product, the hydrolysisproduct comprising glucose, cellobiose, glucose oligomers, or acombination thereof, wherein the feedstock supply line, the upflowhydrolysis reactor, or both the feedstock supply line and the upflowhydrolysis reactor, comprise an enzyme composition comprising cellulaseenzymes and one or more than one flocculent.
 59. The system of claim 58,wherein the feedstock supply line comprises a pre-treated feedstock. 60.The system of claim 58, wherein the solids-liquid separator is selectedfrom the group consisting of a settling tank, a clarifier, a clarifierzone, a centrifuge and a filter.
 61. The system of claim 60, wherein thesolids-liquid separator is a settling tank.
 62. The system of claim 60,wherein the solids-liquid separator is a clarifier zone.
 63. The systemof claim 60, wherein the solids-liquid separator is a filter.
 64. Thesystem of claim 58, wherein the cellulase enzymes are present at adosage from about 1.0 to about 40.0 FPU per gram of cellulose of thepre-treated feedstock.
 65. The system of claim 58, wherein the one ormore than one flocculent is present at a dosage from about 0.1 to about4.0 kg per tonne solids of the pre-treated feedstock. 66-68. (canceled)